PROKARYOTES
crossm Draft Genome Sequence of Zobellia sp. Strain OII3, Isolated from the Coastal Zone of the Baltic Sea Henrik Harms,a,b Anja Poehlein,c Andrea Thürmer,c Gabriele M. König,a,b Till F. Schäberlea,b,d Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germanya; German Center for Infection Research (DZIF) Partner Site Bonn/Cologne, Bonn, Germanyb; Department of Genomics and Applied Microbiology and Göttingen Genomics Laboratory, Georg-August University Göttingen, Göttingen, Germanyc; Institute for Insect Biotechnology, Justus Liebig University Giessen, Giessen, Germanyd
ABSTRACT Zobellia sp. strain OII3 was isolated from a marine environmental sample due to its heterotrophic lifestyle, i.e., using Escherichia coli cells as prey. It shows strong agar-lytic activity. The genome was assembled into 41 contigs with a total size of 5.4 Mb, revealing the genetic basis for natural product biosynthesis.
T
he genus Zobellia contains Gram-negative marine bacteria that show gliding activity (1). Genome sequences were previously available for two strains, i.e., Z. galactanivorans DSM12802 (RefSeq NC_015844) and Z. uliginosa (RefSeq NZ_JQMD00000000). Zobellia spp. have been isolated from marine material, especially algae, and various algae produce galactans like agar and carrageenan. Zobellia spp. seem to be involved in the degradation of algae in the marine environment, and the capability to degrade agar actively is a key feature of this genus, which became a model for studying algal polysaccharide bioconversions (2). The strain described here was isolated during a bioproject aiming to discover novel compounds with biological activities. It was retrieved from marine sediment collected at the coastal zone of Kappeln, Germany, using Escherichia coli cells as prey. The same method was established earlier for terrestrial bacteria, e.g., myxobacteria, which show a high potential for the production of biologically active specialized compounds (3–5). The same strategy proves true for marine samples; in particular, bacteria attached to surfaces of organic material have been isolated in this way. The heterotrophic lifestyle seems to be an advantageous feature for these organisms (5). The ability of Zobellia spp. to degrade agar is particularly pronounced and can be observed by every growing culture on agar plates. The colonies sink into the agar due to the degrading activity. Screening of the extract from Zobellia sp. strain OII3, grown 7 days in ASW-CY medium (casiton 3 g/liter, yeast extract 1 g/liter, KCl 0.99 g/liter, KBr 0.15 g/liter, NaCl 35.22 g/liter, MgCl ⫻ 6 H2O 15.92 g/liter, CaCl2 ⫻ 2 H2O, SrCl ⫻ 6 H2O 0.06 g/liter, Na2SO4 anhydrous 5.88 g/L, NaHCO3 0.29 g/liter, and H3BO3 0.05 g/liter, trace element solution 1 ml/liter, cyanocobalamine stock solution 1 ml/liter; trace element solution: 20 mg ZnCl2, 100 mg MnCl2 ⫻ 4 H2O, 10 mg H3BO3, 10 mg CuSO4, 20 mg CoCl2, 5 mg SnCl2 ⫻ 2 H2O, 5 mg LiCl, 20 mg KBr, 20 mg KI, 10 mg Na2MoO4 ⫻ 2 H2O and 5.2 g Na2-EDTA ⫻ 2 H2O dissolved in 1 liter distilled water; cyanocobalamine stock solution: 0.5 mg dissolved in 1 ml water), revealed antimicrobial activity against Gram-positive test strains. A sample was prepared for sequencing by growing the strain aerobically at 30°C in ASW-CY medium for 3 days. Extraction of the genomic DNA was performed by using the GenElute bacterial genomic DNA kit (Sigma Aldrich). The extracted DNA was used to generate Illumina shotgun paired-end sequencing libraries, which were sequenced Volume 5 Issue 36 e00737-17
Received 27 June 2017 Accepted 3 July 2017 Published 7 September 2017 Citation Harms H, Poehlein A, Thürmer A, König GM, Schäberle TF. 2017. Draft genome sequence of Zobellia sp. strain OII3, isolated from the coastal zone of the Baltic Sea. Genome Announc 5:e00737-17. https://doi .org/10.1128/genomeA.00737-17. Copyright © 2017 Harms et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Gabriele M. König, [email protected], or Till F. Schäberle, [email protected].
genomea.asm.org 1
Harms et al.
with a MiSeq instrument and the MiSeq reagent kit version 3, as recommended by the manufacturer (Illumina). Quality filtering using Trimmomatic version 0.36 (6) resulted in 2,211,244 paired-end reads. The assembly was performed with the SPAdes genome assembler software version 3.10.0 (7). The assembly resulted in 41 contigs (⬎500 bp) and an average coverage of 92.87-fold. The assembly was validated, and the read coverage was determined with QualiMap version 2.1 (8). The resulting draft genome is 5,396,230 bp in length, while the GC content is 42.6%. The sequence was annotated with the Rapid Annotations using Subsystems Technology (RAST) prokaryotic genome annotation server (version 2.0) using standard procedures (9), yielding 4,648 coding sequences. An antiSMASH (10) analysis was performed to identify putative biosynthetic gene clusters. In total, five putative clusters were identified, i.e., those coding for an aryl polyene type III polyketide, resorcinol, two terpenes, and aryl polyene-resorcinol. The same clusters were found in the closest relative strain, Z. galactanivorans DSM12802 (RefSeq NC_015844), which harbored an additional putative type I PKS biosynthetic gene cluster that was not detected by antiSMASH in the strain reported here. The genome of Zobellia sp. OII3 will contribute to the identification of the antibiotically active component(s) of this strain. Accession number(s). This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. MWRZ00000000. The version described in this paper is the first version, MWRZ01000000. ACKNOWLEDGMENTS We thank Mila Goralski for technical assistance. This work was supported by the German Center for Infection Research (DZIF) through grant TTU09.811. Sequencing was performed by the Göttingen Genomics Laboratory (G2L).
REFERENCES 1. Barbeyron T, L’Haridon S, Corre E, Kloareg B, Potin P. 2001. Zobellia galactanovorans gen. nov., sp. nov., a marine species of Flavobacteriaceae isolated from a red alga, and classification of [Cytophaga] uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Zobellia uliginosa gen. nov., comb. nov. Int J Syst Evol Microbiol 51:985–997. https://doi .org/10.1099/00207713-51-3-985. 2. Barbeyron T, Thomas F, Barbe V, Teeling H, Schenowitz C, Dossat C, Goesmann A, Leblanc C, Oliver Glöckner F, Czjzek M, Amann R, Michel G. 2016. Habitat and taxon as driving forces of carbohydrate catabolism in marine heterotrophic bacteria: example of the model algae-associated bacterium Zobellia galactanivorans Dsij T . Environ Microbiol 18: 4610 – 4627. https://doi.org/10.1111/1462-2920.13584. 3. Schäberle TF, Lohr F, Schmitz A, König GM. 2014. Antibiotics from myxobacteria. Nat Prod Rep 31:953–972. https://doi.org/10.1039/c4np00011k. 4. Herrmann J, Fayad AA, Müller R. 2017. Natural products from myxobacteria: novel metabolites and bioactivities. Nat Prod Rep 34: 135–160. https://doi.org/10.1039/c6np00106h. 5. Korp J, Vela Gurovic MS, Nett M. 2016. Antibiotics from predatory bacteria. Beilstein J Org Chem 12:594 – 607. https://doi.org/10.3762/bjoc .12.58. 6. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for
Volume 5 Issue 36 e00737-17
7.
8.
9.
10.
Illumina sequence data. Bioinformatics 30:2114 –2120. https://doi.org/10 .1093/bioinformatics/btu170. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455– 477. https://doi.org/10.1089/cmb.2012.0021. García-Alcalde F, Okonechnikov K, Carbonell J, Cruz LM, Götz S, Tarazona S, Dopazo J, Meyer TF, Conesa A. 2012. Qualimap: evaluating nextgeneration sequencing alignment data. Bioinformatics 28:2678 –2679. https://doi.org/10.1093/bioinformatics/bts503. Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, Edwards RA, Gerdes S, Parrello B, Shukla M, Vonstein V, Wattam AR, Xia F, Stevens R. 2014. The SEED and the rapid annotation of microbial genomes using subsystems technology (RAST). Nucleic Acids Res 42:D206 –DD214. https://doi.org/10.1093/nar/gkt1226. Weber T, Blin K, Duddela S, Krug D, Kim HU, Bruccoleri R, Lee SY, Fischbach MA, Müller R, Wohlleben W, Breitling R, Takano E, Medema MH. 2015. antiSMASH 3.0 —a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res 43:W237–W243. https://doi.org/10.1093/nar/gkv437.
genomea.asm.org 2
crossm Draft Genome Sequence of Zobellia sp. Strain OII3, Isolated from the Coastal Zone of the Baltic Sea Henrik Harms,a,b Anja Poehlein,c Andrea Thürmer,c Gabriele M. König,a,b Till F. Schäberlea,b,d Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germanya; German Center for Infection Research (DZIF) Partner Site Bonn/Cologne, Bonn, Germanyb; Department of Genomics and Applied Microbiology and Göttingen Genomics Laboratory, Georg-August University Göttingen, Göttingen, Germanyc; Institute for Insect Biotechnology, Justus Liebig University Giessen, Giessen, Germanyd
ABSTRACT Zobellia sp. strain OII3 was isolated from a marine environmental sample due to its heterotrophic lifestyle, i.e., using Escherichia coli cells as prey. It shows strong agar-lytic activity. The genome was assembled into 41 contigs with a total size of 5.4 Mb, revealing the genetic basis for natural product biosynthesis.
T
he genus Zobellia contains Gram-negative marine bacteria that show gliding activity (1). Genome sequences were previously available for two strains, i.e., Z. galactanivorans DSM12802 (RefSeq NC_015844) and Z. uliginosa (RefSeq NZ_JQMD00000000). Zobellia spp. have been isolated from marine material, especially algae, and various algae produce galactans like agar and carrageenan. Zobellia spp. seem to be involved in the degradation of algae in the marine environment, and the capability to degrade agar actively is a key feature of this genus, which became a model for studying algal polysaccharide bioconversions (2). The strain described here was isolated during a bioproject aiming to discover novel compounds with biological activities. It was retrieved from marine sediment collected at the coastal zone of Kappeln, Germany, using Escherichia coli cells as prey. The same method was established earlier for terrestrial bacteria, e.g., myxobacteria, which show a high potential for the production of biologically active specialized compounds (3–5). The same strategy proves true for marine samples; in particular, bacteria attached to surfaces of organic material have been isolated in this way. The heterotrophic lifestyle seems to be an advantageous feature for these organisms (5). The ability of Zobellia spp. to degrade agar is particularly pronounced and can be observed by every growing culture on agar plates. The colonies sink into the agar due to the degrading activity. Screening of the extract from Zobellia sp. strain OII3, grown 7 days in ASW-CY medium (casiton 3 g/liter, yeast extract 1 g/liter, KCl 0.99 g/liter, KBr 0.15 g/liter, NaCl 35.22 g/liter, MgCl ⫻ 6 H2O 15.92 g/liter, CaCl2 ⫻ 2 H2O, SrCl ⫻ 6 H2O 0.06 g/liter, Na2SO4 anhydrous 5.88 g/L, NaHCO3 0.29 g/liter, and H3BO3 0.05 g/liter, trace element solution 1 ml/liter, cyanocobalamine stock solution 1 ml/liter; trace element solution: 20 mg ZnCl2, 100 mg MnCl2 ⫻ 4 H2O, 10 mg H3BO3, 10 mg CuSO4, 20 mg CoCl2, 5 mg SnCl2 ⫻ 2 H2O, 5 mg LiCl, 20 mg KBr, 20 mg KI, 10 mg Na2MoO4 ⫻ 2 H2O and 5.2 g Na2-EDTA ⫻ 2 H2O dissolved in 1 liter distilled water; cyanocobalamine stock solution: 0.5 mg dissolved in 1 ml water), revealed antimicrobial activity against Gram-positive test strains. A sample was prepared for sequencing by growing the strain aerobically at 30°C in ASW-CY medium for 3 days. Extraction of the genomic DNA was performed by using the GenElute bacterial genomic DNA kit (Sigma Aldrich). The extracted DNA was used to generate Illumina shotgun paired-end sequencing libraries, which were sequenced Volume 5 Issue 36 e00737-17
Received 27 June 2017 Accepted 3 July 2017 Published 7 September 2017 Citation Harms H, Poehlein A, Thürmer A, König GM, Schäberle TF. 2017. Draft genome sequence of Zobellia sp. strain OII3, isolated from the coastal zone of the Baltic Sea. Genome Announc 5:e00737-17. https://doi .org/10.1128/genomeA.00737-17. Copyright © 2017 Harms et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Gabriele M. König, [email protected], or Till F. Schäberle, [email protected].
genomea.asm.org 1
Harms et al.
with a MiSeq instrument and the MiSeq reagent kit version 3, as recommended by the manufacturer (Illumina). Quality filtering using Trimmomatic version 0.36 (6) resulted in 2,211,244 paired-end reads. The assembly was performed with the SPAdes genome assembler software version 3.10.0 (7). The assembly resulted in 41 contigs (⬎500 bp) and an average coverage of 92.87-fold. The assembly was validated, and the read coverage was determined with QualiMap version 2.1 (8). The resulting draft genome is 5,396,230 bp in length, while the GC content is 42.6%. The sequence was annotated with the Rapid Annotations using Subsystems Technology (RAST) prokaryotic genome annotation server (version 2.0) using standard procedures (9), yielding 4,648 coding sequences. An antiSMASH (10) analysis was performed to identify putative biosynthetic gene clusters. In total, five putative clusters were identified, i.e., those coding for an aryl polyene type III polyketide, resorcinol, two terpenes, and aryl polyene-resorcinol. The same clusters were found in the closest relative strain, Z. galactanivorans DSM12802 (RefSeq NC_015844), which harbored an additional putative type I PKS biosynthetic gene cluster that was not detected by antiSMASH in the strain reported here. The genome of Zobellia sp. OII3 will contribute to the identification of the antibiotically active component(s) of this strain. Accession number(s). This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. MWRZ00000000. The version described in this paper is the first version, MWRZ01000000. ACKNOWLEDGMENTS We thank Mila Goralski for technical assistance. This work was supported by the German Center for Infection Research (DZIF) through grant TTU09.811. Sequencing was performed by the Göttingen Genomics Laboratory (G2L).
REFERENCES 1. Barbeyron T, L’Haridon S, Corre E, Kloareg B, Potin P. 2001. Zobellia galactanovorans gen. nov., sp. nov., a marine species of Flavobacteriaceae isolated from a red alga, and classification of [Cytophaga] uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Zobellia uliginosa gen. nov., comb. nov. Int J Syst Evol Microbiol 51:985–997. https://doi .org/10.1099/00207713-51-3-985. 2. Barbeyron T, Thomas F, Barbe V, Teeling H, Schenowitz C, Dossat C, Goesmann A, Leblanc C, Oliver Glöckner F, Czjzek M, Amann R, Michel G. 2016. Habitat and taxon as driving forces of carbohydrate catabolism in marine heterotrophic bacteria: example of the model algae-associated bacterium Zobellia galactanivorans Dsij T . Environ Microbiol 18: 4610 – 4627. https://doi.org/10.1111/1462-2920.13584. 3. Schäberle TF, Lohr F, Schmitz A, König GM. 2014. Antibiotics from myxobacteria. Nat Prod Rep 31:953–972. https://doi.org/10.1039/c4np00011k. 4. Herrmann J, Fayad AA, Müller R. 2017. Natural products from myxobacteria: novel metabolites and bioactivities. Nat Prod Rep 34: 135–160. https://doi.org/10.1039/c6np00106h. 5. Korp J, Vela Gurovic MS, Nett M. 2016. Antibiotics from predatory bacteria. Beilstein J Org Chem 12:594 – 607. https://doi.org/10.3762/bjoc .12.58. 6. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for
Volume 5 Issue 36 e00737-17
7.
8.
9.
10.
Illumina sequence data. Bioinformatics 30:2114 –2120. https://doi.org/10 .1093/bioinformatics/btu170. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455– 477. https://doi.org/10.1089/cmb.2012.0021. García-Alcalde F, Okonechnikov K, Carbonell J, Cruz LM, Götz S, Tarazona S, Dopazo J, Meyer TF, Conesa A. 2012. Qualimap: evaluating nextgeneration sequencing alignment data. Bioinformatics 28:2678 –2679. https://doi.org/10.1093/bioinformatics/bts503. Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, Edwards RA, Gerdes S, Parrello B, Shukla M, Vonstein V, Wattam AR, Xia F, Stevens R. 2014. The SEED and the rapid annotation of microbial genomes using subsystems technology (RAST). Nucleic Acids Res 42:D206 –DD214. https://doi.org/10.1093/nar/gkt1226. Weber T, Blin K, Duddela S, Krug D, Kim HU, Bruccoleri R, Lee SY, Fischbach MA, Müller R, Wohlleben W, Breitling R, Takano E, Medema MH. 2015. antiSMASH 3.0 —a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res 43:W237–W243. https://doi.org/10.1093/nar/gkv437.
genomea.asm.org 2
